Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A.

Wilforlide A is one of the main active constituents produced in trace amounts in Tripterygium wilfordii Hook F, which has excellent anti-inflammatory and immune suppressive effects. Despite the seeming structural simplicity of the compound, the biosynthetic pathway of wilforlide A remains unknown. Gene-specific expression analysis and genome mining were used to identify the gene candidates, and their functions were studied in vitro and in vivo. A modularized two-step (M2S) technique and CRISPR-Cas9 methods were used to construct engineering yeast. Here, we identified a cytochrome P450, TwCYP82AS1, that catalyses C-22 hydroxylation during wilforlide A biosynthesis. We also found that TwCYP712K1 to K3 can further oxidize the C-29 carboxylation of oleanane-type triterpenes in addition to friedelane-type triterpenes. Reconstitution of the biosynthetic pathway in engineered yeast increased the precursor supply, and combining TwCYP82AS1 and TwCYP712Ks produced abrusgenic acid, which was briefly acidified to achieve the semisynthesis of wilforlide A. Our work presents an alternative metabolic engineering approach for obtaining wilforlide A without relying on extraction from plants.

Characterization of a Xylosyltransferase from Panax notoginseng Catalyzing Ginsenoside 2'-O Glycosylation in the Biosynthesis of Notoginsenosides.

Notoginsenosides are important bioactive compounds from Panax notoginseng (Burk.) F. H. Chen, most of which have xylose in their sugar chains. However, the xylosyltransferases involved in the generation of notoginsenosides remain poorly understood, posing a bottleneck for further study of the biosynthesis of notoginsenosides. In this work, a new xylosyltransferase gene, PnUGT57 (named UGT94BW1), was identified from P. notoginseng, which has a distinct sequence and could catalyze the 2'-O glycosylation of ginsenosides Rh1 and Rg1 to produce notoginsenosides R2 and R1, respectively. We first characterized the optimal conditions for the PnUGT57 activity and its enzymatic kinetic parameters, and then, molecular docking and site-directed mutagenesis were performed to elucidate the catalytic mechanism of PnUGT57. Combined with the results of site-directed mutagenesis, Glu26, Ser266, Glu267, Trp347, Ser348, and Glu352 in PnUGT57 were identified as the key residues involved in 2'-O glycosylation of C-6 O-Glc, and PnUGT57R175A and PnUGT57G237A could significantly improve the catalytic activity of PnUGT57. These findings not only provide a new xylosyltransferase gene for augmenting the plant xylosyltransferase database but also identify the pivotal sites and catalytic mechanism of the enzyme, which would provide reference for the modification and application of xylosyltransferases in the future.

Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae.

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L-1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

Novel biocompatible N-rich AIE fluorescent probe for live cell imaging and visual onsite detection of uranium.

A sensitive and biocompatible N-rich probe for rapid visual uranium detection was constructed by grafting two trianiline groups to 2,6-bis(aminomethyl)pyridine. Possessing excellent aggregation-induced emission (AIE) property and the advantages to form multidentate chelate with U selectively, the probe has been applied successfully to visualize uranium in complex environmental water samples and living cells, demonstrating outstanding anti-interference ability against large equivalent of different ions over a wide effective pH range. A large linear range (1.0 × 10-7-9.0 × 10-7 mol/L) and low detection limit (72.6 nmol/L, 17.28 ppb) were achieved for the visual determination of uranium. The recognition mechanism, photophysical properties, analytical performance and cytotoxicity were systematically investigated, demonstrating high potential for fast risk assessment of uranium pollution in field and in vivo.

Linking Daily Victimization to Daily Affect Among Adolescents: The Mediating Role of Sleep Quality and Disturbance.

Bullying victimization is prevalent among adolescents and often linked to emotional problems. Prior studies have been focused on the concurrent or longitudinal associations between bullying victimization and emotional problems, but the daily associations and the underlying mechanisms remain unclear. Implementing daily diary method, the study aimed to examine the links between daily victimization and positive and negative affect as well as the mediating role of sleep quality and disturbance. A total of 265 Chinese adolescents (Mage = 11.65, SD = 0.74; 32.80% females) participated in this study and completed 7-day daily diaries on bullying victimization (traditional and cyber victimization), sleep quality and disturbance, and affect. As hypothesized, at the between-person level, sleep disturbance mediated the relationships between both traditional and cyber victimization and subsequent negative affect. At the within-person level, sleep quality mediated the pathway between traditional victimization and next-day negative affect; furthermore, sleep disturbance mediated the pathway between traditional victimization and positive affect the following day. These findings highlight the mediating roles of sleep quality and sleep disturbance in the relationships between stressful victimizing experiences and emotional problems and also provide novel insights into these associations.

Thy-1 knockdown promotes the osteogenic differentiation of GMSCs via the Wnt/ß-catenin pathway.

Gingival mesenchymal stem cells (GMSCs) are newly developed seed cells for tissue engineering owing to their easy isolation, abundance and high growth rates. Thy-1 is an important regulatory molecule in the differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the function of Thy-1 in the osteogenic differentiation of GMSCs by reducing the expression of Thy-1 using a lentivirus. The results demonstrated that Thy-1 knockdown promoted the osteogenic differentiation of GMSCs in vitro. Validation by RNA-seq revealed an obvious decrease in Vcam1 and Sox9 gene expression with Thy-1 knockdown. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed genes were enriched in the Wnt signalling pathway. We further demonstrated that Thy-1 knockdown promoted osteogenic differentiation of GMSCs by activating the Wnt/ß-catenin signalling pathway. Therefore, Thy-1 has a key regulatory role in the differentiation of GMSCs and maybe a core molecule connecting transcription factors related to the differentiation of MSCs. Our study also highlighted the potential of Thy-1 to modify MSCs, which may help improve their use in tissue engineering.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

Environmental noise exposure and health outcomes: an umbrella review of systematic reviews and meta-analysis.

BACKGROUND: Environmental noise is becoming increasingly recognized as an urgent public health problem, but the quality of current studies needs to be assessed. To evaluate the significance, validity and potential biases of the associations between environmental noise exposure and health outcomes. METHODS: We conducted an umbrella review of the evidence across meta-analyses of environmental noise exposure and any health outcomes. A systematic search was done until November 2021. PubMed, Cochrane, Scopus, Web of Science, Embase and references of eligible studies were searched. Quality was assessed by AMSTAR and Grading of Recommendations, Assessment, Development and Evaluation (GRADE). RESULTS: Of the 31 unique health outcomes identified in 23 systematic reviews and meta-analyses, environmental noise exposure was more likely to result in a series of adverse outcomes. Five percent were moderate in methodology quality, the rest were low to very low and the majority of GRADE evidence was graded as low or even lower. The group with occupational noise exposure had the largest risk increment of speech frequency [relative risk (RR): 6.68; 95% confidence interval (CI): 3.41-13.07] and high-frequency (RR: 4.46; 95% CI: 2.80-7.11) noise-induced hearing loss. High noise exposure from different sources was associated with an increased risk of cardiovascular disease (34%) and its mortality (12%), elevated blood pressure (58-72%), diabetes (23%) and adverse reproductive outcomes (22-43%). In addition, the dose-response relationship revealed that the risk of diabetes, ischemic heart disease (IHD), cardiovascular (CV) mortality, stroke, anxiety and depression increases with increasing noise exposure. CONCLUSIONS: Adverse associations were found for CV disease and mortality, diabetes, hearing impairment, neurological disorders and adverse reproductive outcomes with environmental noise exposure in humans, especially occupational noise. The studies mostly showed low quality and more high-quality longitudinal study designs are needed for further validation in the future.

Draft Genome and Biological Characteristics of Fusarium solani and Fusarium oxysporum Causing Black Rot in Gastrodia elata.

Gastrodia elata is a valuable traditional Chinese medicinal plant. However, G. elata crops are affected by major diseases, such as brown rot. Previous studies have shown that brown rot is caused by Fusarium oxysporum and F. solani. To further understand the disease, we studied the biological and genome characteristics of these pathogenic fungi. Here, we found that the optimum growth temperature and pH of F. oxysporum (strain QK8) and F. solani (strain SX13) were 28 °C and pH 7, and 30 °C and pH 9, respectively. An indoor virulence test showed that oxime tebuconazole, tebuconazole, and tetramycin had significant bacteriostatic effects on the two Fusarium species. The genomes of QK8 and SX13 were assembled, and it was found that there was a certain gap in the size of the two fungi. The size of strain QK8 was 51,204,719 bp and that of strain SX13 was 55,171,989 bp. Afterwards, through phylogenetic analysis, it was found that strain QK8 was closely related to F. oxysporum, while strain SX13 was closely related to F. solani. Compared with the published whole-genome data for these two Fusarium strains, the genome information obtained here is more complete; the assembly and splicing reach the chromosome level. The biological characteristics and genomic information we provide here lay the foundation for further research on G. elata brown rot.

Effects of Dietary Protein-to-Energy Ratios on Growth, Immune Response, Antioxidative Capacity, Liver and Intestinal Histology, and Growth-Related Gene Expression in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂).

This study is aimed at evaluating the effects of dietary protein-to-energy ratios on the growth, immunological response, antioxidative capacity, liver and intestinal histology, and growth-related gene expression of hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂). Eight diets were formulated to form different protein/energy ratios of 84, 88, 90, 93, 95, 96, 99, and 103 mg/kcal (P/E84, P/E88, P/E90, P/E93, P/E95, P/E96, P/E99, and P/E103), respectively. These diets contain different levels of gross energy (GE), ranging from 4.13 to 4.76 kcal g-1. Seven hundred and twenty healthy fish (17.15 ± 0.02 g) were randomly dispersed into 24 rectangular fiberglass tanks with 8 treatments in triplicate groups. The fish fed a P/E ratio of 95 mg/kcal demonstrated the best growth and feed utilization. A significant (P < 0.05) increase in percent weight gain (WG%) and specific growth rate (SGR) was seen as the dietary P/E ratio ameliorated from P/E84 to P/E95, followed by a decreased pattern in these parameters. Feed conversion ratio (FCR) and daily feed intake (DFI) were significantly impacted by dietary P/E ratios (P < 0.05). Additionally, an optimum P/E ratio improved intestinal morphology. However, low or high P/E ratio diets can cause oxidative stress, impaired liver function, and significantly reduced nonspecific immunity. The expression of target of rapamycin (TOR) and insulin-like growth factor-1 (IGF1) genes in the liver was considerably influenced by dietary protein-to-energy ratios (P < 0.05). Based on the statistical analysis of WG% against the dietary P/E ratio, the optimal P/E ratio for the studied species was estimated to be 92.92 mg/kcal.

The Optimum Dietary Phenylalanine Requirement of Hybrid Grouper (Epinephelusfuscoguttatus ♀ × Epinepheluslanceolatus ♂) Juveniles: Effects on Growth Performance, Gut Micromorphology, and Antioxidation.

The optimum phenylalanine (Phe) requirement for hybrid grouper (Epinephelusfuscoguttatus ♀ × Epinepheluslanceolatus ♂) juveniles was determined through an 8-week growth trial. A total of seven isoenergetic (340 kcal per 100 g of dry matter), isonitrogenous, and isolipidic diets were made, containing 8.2 (Phe 8.2), 9.2 (Phe 9.2), 10.1 (Phe 10.1), 11.2 (Phe 11.2), 13.3 (Phe 13.3), 15.2 (Phe 15.2), and 17.3 g/kg (Phe 17.3), respectively. Triplicate tanks of juvenile fish (about 16.7 g/fish) were fed each experimental diet twice daily until apparent satiation. The results indicated that different dietary Phe levels significantly influenced weight gain percentage (WG), feed efficiency (FE), protein efficiency ratio (PER), as well as, productive protein value (PPV). Fish fed Phe 8.2 had the lowest WG or PPV among all experimental treatments. Furthermore, the optimal dietary Phe level increased fold height, width, enterocyte, and microvillus height of fish. The Phe 10.1 group exhibited higher growth hormone (GH) expression in the pituitary compared to other groups. Expression of hepatic insulin-like growth factor-1 (IGF-1) and growth hormone receptor 1 (GHR1) displayed a similar pattern of variation to that of GH. The Phe 13.3 group had lower expression of S6 kinase 1 (S6K1) and target of rapamycin (TOR) than other groups. In addition, fish fed Phe 10.1 had lower levels of nuclear factor erythroid 2 (Nrf2) and heat shock protein 70 (HSP70) in the head kidney, and Cu/Zn-superoxide (Cu/ZnSOD) dismutases in the midgut compared to fish fed other Phe levels. Generally, optimal Phe content in the diet of hybrid grouper was estimated to be 12.7 g/kg of dry matter (27.3 g/kg of dietary protein), and at this level, the feed utilization, gut micromorphology, and immunity of fish were also elevated.

[Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius].

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 µmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

Identification of the metabolites of methylophiopogonanone A by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry.

RATIONALE: Methylophiopogonanone A (MOA) is a naturally occurring homoisoflavonoid from the Chinese herb Ophiopogon japonicus, which has been demonstrated to attenuate myocardial apoptosis. However, the metabolism of MOA remains unknown. The goal of the present work was to investigate the in vitro metabolism of MOA using liver microsomes and hepatocytes. METHODS: The metabolites were generated by incubating MOA with rat, monkey and human liver microsomes or hepatocytes. The resulting samples were analyzed by using a quadrupole-orbitrap high-resolution mass spectrometer. The metabolites were identified through the measurements of the exact mass, elemental composition and product ions. RESULTS: A total of 15 metabolites were detected and identified. Among these metabolites, M7 (demethylenation) was the most abundant metabolite in liver microsomes, while M6 (hydroxylation) was the predominant metabolite in hepatocytes, and glucuronidation metabolites (M9 and M10) were also the main metabolites in hepatocytes. The metabolic pathways of MOA included hydroxylation, demethylenation, glucuronidation, methylation, sulfation and glutathione conjugation. CONCLUSIONS: This study for the first time provides valuable data on the metabolites of MOA, which will be of great importance for a better understanding of its disposition and to predict human pharmacokinetics.

A novel fluorescence ratio probe based on dual-emission carbon dots for highly selective and sensitive detection of chlortetracycline and cell imaging.

The novel dual-emission carbon dots (DECDs) for highly selective and sensitive recognition of chlortetracycline (CTC) and cell imaging were synthesized successfully by one-step synthesis. The obtained DECDs possessed two fluorescence peaks (345 nm and 450 nm) and showed specific response to CTC, resulting in a decrease in fluorescence intensity at 345 nm, a blue shift, and an increase in fluorescence intensity at 450 nm. The obtained DECDs exhibited highly selective response to CTC and not to its analogues, such as tetracycline, doxycycline, and oxytetracycline. Thus, an excellent ratiometric probe for the detection of CTC was fabricated successfully and used for the detection of CTC in real samples with the detection limit (LOD) of 16.45 nM. More importantly, the DECDs were used for quantitative detection of CTC in living cells, which demonstrated excellent biocompatibility and broad prospects in biomedicine application. Finally, the excellent selectivity of DECDs toward CTC was attributed to the FRET mechanism and the formation of complexes.

Analysis of droplet size uniformity and selection of spray parameters based on the biological optimum particle size theory.

Based on the theory of biological optimal particle size, the most easily attached droplets for different organisms have different particle sizes. To achieve the best average particle size, the droplet size in the atomization field must be more uniform and attain a high the adhesion rate. Therefore, during the application process, not only the average particle size of the droplets but also the influence of the uniformity of the droplets in the spray field must be considered. In this study, 20 small-angle fan nozzles ranging from 20° to 40° are used as the research objects. The droplet size information in the atomization field is obtained using a laser particle size analyzer, and the droplet uniformity under different parameters is calculated. The results showed that within the range of the parameters selected in the experiment, the droplet size increased with an increase in the flow rate, and decreased with an increase in the pressure. In addition, the angle had little effect on the droplet size. Increasing the spray height, spray angle, and pressure, while reducing the equivalent outlet diameter of the nozzle was beneficial to improve the uniformity of droplets. The order of the degree of influence of the four parameters on the uniformity of the droplets was height > equivalent outlet diameter (r) > pressure > spray angle, and the influence weights were 51.1%, 37.1%, 7.8%, 4.1%; 48.4%, 37.6%, 10%, and 4%. Under the condition of the parameter settings used in this experiment, the optimal atomization effect for the four intervals of 150-200 µm, 200-250 µm, 250-300 µm, and 300-400 µm was analyzed from the perspective of uniformity. The nozzle models with the best atomization effects in each interval were SS4003-0.3 MPa, SS4006-0.3 MPa, SS4008-0.3 MPa, and 633.512.30.CC-0.1 MPa.

Skeletal Maturation in Patients With Cleft Lip and/or Palate: A Systematic Review.

OBJECTIVE: The objective of this systematic review was to evaluate the evidence regarding skeletal maturation in patients with cleft lip and/or palate (CL/P) and to investigate whether the skeletal maturation is delayed in these patients. DESIGN: Systematic review. METHODS: Electronic and manual searches of scientific literature were conducted in 4 databases (MEDLINE, Embase, Cochrane Library, and Web of Science). Cohort studies that compared the skeletal maturation of patients with CL/P with that of children without CL/P were eligible for inclusion. The quality of included cohort studies was assessed using the Newcastle-Ottawa Scale. PATIENTS AND PARTICIPANTS: Patients of any sex and ethnicity with CL/P and children without CL/P were included in this systematic review. MAIN OUTCOME MEASURES: Difference in skeletal maturation between patients with CL/P and patients without CL/P. RESULTS: Thirteen retrospective cohort studies were included in this systematic review. Ten studies were considered of high quality and 3 were considered of general quality. The results of the included studies comparing skeletal maturation of patients with CL/P and children without CL/P were heterogeneous. CONCLUSION: Heterogeneity of skeletal maturation assessment methods, chronological age, sex, cleft type, and race may influence the final results of clinical studies on skeletal maturation in patients with CL/P. Overall, there is limited evidence to determine whether the skeletal maturation level of patients with CL/P is delayed compared to that of normal children. Further studies are needed to determine the skeletal maturation patterns in patients with CL/P.

A bean common mosaic virus-resistance gene in the soybean variant V94-5152 was mapped to the Rsv4 locus conferring resistance to soybean mosaic virus.

KEY MESSAGE: In the soybean variant V94-5152, a BCMV-resistance gene was mapped near to the region of SMV-resistance Rsv4 locus, raising a possibility that V94-5152 may rely on Rsv4 locus to resist against both SMV and BCMV. Both Soybean mosaic virus (SMV) and Bean common mosaic virus (BCMV) can induce soybean mosaic diseases, but few studies have explored soybean resistance against BCMV so far. In this study, V94-5152, a soybean variant resistant to BCMV and SMV, was crossed with a susceptible cultivar, Williams 82 to map the resistance gene. By inoculating 292 F2 individuals with a BCMV isolate HZZB011, a segregation ratio of 3 resistant: 1 susceptible was observed, suggesting that V94-5152 possesses a single-dominant resistance gene against BCMV-HZZB011. Bulk segregation analysis (BSA) then revealed that the resistance gene is closely linked to BARCSOYSSR_02_0617, a simple sequence repeat (SSR) marker on chromosome 2. Genotyping neighboring SSR markers among the 292 F2 individuals enabled us to draw a genetic linkage map, which indicated that the BCMV-resistance gene is located 0.2 cM downstream of BARCSOYSSR_02_0617. Amplification and sequencing ten candidate genes (Glyma02g121300 to Glyma02g122200) around this marker then revealed four genes containing nonsynonymous changes or indels. Also, this location is near to the recently cloned SMV-resistance Rsv4 locus from the cultivar Peking. By obtaining ten more sequences of Rsv4 locus from cultivated and wild soybean materials, we further investigated the variation and evolutionary patterns of this virus-resistance locus. It was evident that positive selections had been acting on this locus, with one critical amino acid change (R55P) shared by all resistance soybeans tested.

Dietary valine levels affect growth, protein utilisation, immunity and antioxidant status in juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

A 6-week growth trial was conducted to evaluate the influences of dietary valine (Val) levels on growth, protein utilisation, immunity, antioxidant status and gut micromorphology of juvenile hybrid groupers. Seven isoenergetic, isoproteic and isolipidic diets were formulated to contain graded Val levels (1·21, 1·32, 1·45, 1·58, 1·69, 1·82 and 1·94 %, DM basis). Each experimental diet was hand-fed to triplicate groups of twelve hybrid grouper juveniles. Results showed that weight gain percentage (WG%), protein productive value, protein efficiency ratio, and feed efficiency were increased as dietary Val level increased, reaching a peak value at 1·58 % dietary Val. The quadratic regression analysis of WG% against dietary Val levels indicated that the optimum dietary Val requirement for hybrid groupers was estimated to be 1·56 %. Gut micromorphology and expression of growth hormone in pituitary, insulin-like growth factor 1, target of rapamycin and S6 kinase 1 in liver were significantly affected by dietary Val levels. In serum, fish fed 1·58 % dietary Val had higher superoxide dismutase, catalase, lysozyme activities and IgM concentrations than fish fed other dietary Val levels. Fish fed 1·58 % dietary Val had higher expression of NF-E2-related factor 2 in head kidney than fish fed other dietary Val levels. Generally, the optimum dietary Val requirement for maximal growth of hybrid groupers was estimated to be 1·56 % of DM, corresponding to 3·16 % of dietary protein, and dietary Val levels affected growth, protein utilisation, immunity and antioxidant status in hybrid groupers.

The IGF-1/GH-GLUTs-plasma glucose regulating axis in hybrid grouper (Epinephelus fuscoguttatus♀ × epinephelus lanceolatus♂) fed a high-carbohydrate diet.

The carnivorous teleost fish is often intolerant to high levels of postprandial plasma glucose. This study aimed to evaluate the effects of insulin-like growth factor-1 (IGF-1) and growth hormone (GH) administrations on plasma glucose levels and expression of glucose transporters (GLUTs) in various tissues of hybrid grouper, and hence to further clarify the hormone-GLUTs-plasma glucose regulating axis. Twenty-four experimental fish (average body weight: 77.5 ± 5.4 g) were selected and injected with recombinant human IGF-1 (0.2 µg/g body weight) and PBS (0.01 mol/L) in enterocoelia, respectively, and in the GH injected experiment, the same quantity of fish (average body weight: 103.8 ± 5.8 g) were administrated with GH at a dose of 0.5 µg/g body weight or with PBS at a dose of 0.01 mol/L. Results showed that plasma glucose level was significantly (P < 0.05) declined by the IGF-1 administration but elevated by the GH administration. Plasma IGF-1 concentration was significantly (P < 0.01) elevated by the IGF-1 administration, while GH concentration did not significantly (P ≥ 0.05) respond to the GH administration. The relative mRNA levels of insulin-like growth factor-1 receptor a (IGF-Ra) in liver and muscle were decreased significantly with the IGF-1 administration, and a similar variation tendency was also found in insulin-like growth factor-1 receptor b (IGF-Rb) in liver, muscle and adipose tissues. Besides, the relative mRNA level of insulin receptor (IRS) in liver was significantly increased in the IGF-1 administrated group. After the GH administration, the mRNA levels of hepatic growth factor receptor 2 (GHR2) and IGF-1 were significantly elevated. As for GLUTs, the relative mRNA levels of GLUT1 and GLUT2 in liver were obviously elevated by the IGF-1 administration, while the mRNA level of GLUT4 in muscle was reduced. In liver, the protein levels of GLUT1, 2 and 4 were significantly elevated by the IGF-1 administration, and in adipose, only GLUT1 was observed to have a significantly increased protein level. The mRNA expression of GLUTs was less affected by the GH administration. The protein level of GLUT1 in liver was significantly reduced by the GH administration, while in adipose, it was significantly increased. The protein level of GLUT2 in liver or adipose showed an opposite variation as that of GLUT1. Overall, IGF-1 had a hypoglycemic effect on hybrid grouper, and this probably was through up-regulating the protein levels of hepatic GLUT1, 2 and 4 and adipose GLUT1. GH showed an opposite role in regulating plasma glucose level as IGF-1.